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1. Click Conjugation of Boron Dipyrromethene (BODIPY) Fluorophores to EGFR-Targeting Linear and Cyclic Peptides

   https://doi.org/10.3390/molecules26030593  

   Williams, Tyrslai M. ; Kaufman, Nichole E. ; Zhou, Zehua ; Singh, Sitanshu S. ; Jois, Seetharama D. ; Vicente, Maria da ( February 2021 , Molecules)

   null (Ed.)

   Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their ability for binding to EGFR was investigated. The peptide ligands K(N3)LARLLT and its cyclic analog cyclo(K(N3)larllt, previously shown to have high affinity for binding to the extracellular domain of EGFR, were conjugated to alkynyl-functionalized BODIPY dyes 1 and 2 via a copper-catalyzed click reaction. This reaction produced conjugates 3, 4, and 5 in high yields (70–82%). In vitro studies using human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly for the cyclic peptide conjugate 5, and low cytotoxicity in light (~1 J·cm−2) and darkness. Surface plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for EGFR, particularly for 5 bearing the cyclic peptide. Competitive binding studies using three cell lines with different expressions of EGFR show that 5 binds specifically to EGFR-overexpressing colon cancer cells. Among the three conjugates, 5 bearing the cyclic peptide exhibited the highest affinity for binding to the EGFR protein. 

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2. Interactions between heparin and SARS-CoV-2 spike glycoprotein RBD from omicron and other variants

   https://doi.org/10.3389/fmolb.2022.912887  

   Gelbach, Adrianne L. ; Zhang, Fuming ; Kwon, Seok-Joon ; Bates, John T. ; Farmer, Andrew P. ; Dordick, Jonathan S. ; Wang, Chunyu ; Linhardt, Robert J. ( August 2022 , Frontiers in Molecular Biosciences)

   Heparan sulfate (HS) acts as a co-receptor of angiotensin-converting enzyme 2 (ACE2) by interacting with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein (SGP) facilitating host cell entry of SARS-CoV-2 virus. Heparin, a highly sulfated version of heparan sulfate (HS), interacts with a variety of proteins playing key roles in many physiological and pathological processes. In this study, SARS-CoV-2 SGP receptor binding domain (RBD) wild type (WT), Delta and Omicron variants were expressed in Expi293F cells and used in the kinetic and structural analysis on their interactions with heparin. Surface plasmon resonance (SPR) analysis showed the binding kinetics of SGP RBD from WT and Delta variants were very similar while Omicron variant SGP showed a much higher association rate. The SGP from Delta and Omicron showed higher affinity ( K D ) to heparin than the WT SGP. Competition SPR studies using heparin oligosaccharides indicated that binding of SGP RBDs to heparin requires chain length greater than 18. Chemically modified heparin derivatives all showed reduced interactions in competition assays suggesting that all the sulfo groups in the heparin polysaccharide were critical for binding SGP RBDs with heparin. These interactions with heparin are pH sensitive. Acidic pH (pH 6.5, 5.5, 4.5) greatly increased the binding of WT and Delta SGP RBDs to heparin, while acidic pH slightly reduced the binding of Omicron SGP RBD to heparin compared to binding at pH 7.3. In contrast, basic pH (pH 8.5) greatly reduced the binding of Omicron SGP RBDs to heparin, with much less effects on WT or Delta. The pH dependence indicates different charged residues were present at the Omicron SGP-heparin interface. Detailed kinetic and structural analysis of the interactions of SARS-CoV-2 SGP RBDs with heparin provides important information for designing anti-SARS-CoV-2 molecules. 

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3. The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors

   https://doi.org/10.3390/polym14020241  

   Chiodi, Elisa ; Marn, Allison M. ; Bakhshpour, Monireh ; Lortlar Ünlü, Nese ; Ünlü, M. Selim ( January 2022 , Polymers)

   The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches. 

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4. Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

   https://doi.org/10.3390/s21082634  

   Chiodi, Elisa ; Daaboul, George G. ; Marn, Allison M. ; Ünlü, M. Selim ( April 2021 , Sensors)

   null (Ed.)

   Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo. 

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5. Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL

   https://doi.org/10.1128/JVI.00983-20  

   Cairns, Tina M. ; Atanasiu, Doina ; Saw, Wan Ting ; Lou, Huan ; Whitbeck, J. Charles ; Ditto, Noah T. ; Bruun, Birgitte ; Browne, Helena ; Bennett, Lucas ; Wu, Chun ; et al ( September 2020 , Journal of Virology)

   Longnecker, Richard M. (Ed.)

   ABSTRACT A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, “stabilizing” MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV. IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development. 

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